The major accomplishments for this year are summarized below. 1) Engineered antibody domains (eAds) have emerged as a novel class of HIV-1 inhibitors and are currently under preclinical testing as promising drug candidates for prevention and therapy of HIV-1 infection. Reverse mutation of antibodies to germline sequences (germlining) could not only identify less mutated variants with lower probability of immunogenicity and other improved properties but also help elucidate their mechanisms of action. We sequentially reverted the framework (FRs) and complementary determining regions (CDRs) of m36, a human antibody heavy chain variable domain-based eAd targeting the coreceptor binding site of the viral envelope glycoprotein gp120, back to germline sequences. Two types of amino acid mutations and one region in the antibody V segment were identified that are critical for HIV-1 neutralization. These include four mutations to acidic acid residues distributed in the CDR1 and CDR2, two mutations to hydrophobic residues in the FR3 and CDR3, and partial FR2 and FR3 sequences flanking the CDR2 that are derived from a different gene family. An m36 variant with all five mutations in the FRs reverted back to germline showed slightly increased neutralizing activity against two HIV-1 isolates tested. Another variant with seven of twelve mutations in the V segment reverted retained potency within threefold of that of the mature antibody. These results, together with an analysis of m36-gp120-CD4 docking structures, could have implications for the further development of m36 and elucidation of its mechanism of potent and broad HIV-1 neutralization. 2) A fundamental challenge for developing an effective and safe HIV-1 vaccine is to identify vaccine immunogens that can initiate and maintain immune responses leading to elicitation of broadly neutralizing HIV-1 antibodies (bnAbs) through complex maturation pathways. We have previously found that HIV-1 envelope glycoproteins (Env) lack measurable binding to putative germline predecessors of known bnAbs and proposed to search for non-HIV immunogens that could initiate their somatic maturation. Using bnAb b12 as a model bnAb and yeast display technology, we isolated five (poly)peptides from plant leaves, insects, E. coli strains, and sea water microbes that bind to b12 putative germline and intermediate antibodies. Rabbit immunization with the (poly)peptides alone induced high titers of cross-reactive antibodies that neutralized HIV-1 isolates SF162 and JRFL. Priming rabbits with the (poly)peptides followed by boosts with trimeric gp140SF162 and then resurfaced Env (RSC3) induced antibodies that competed with mature b12 and neutralized tier 1 and 2 viruses from clade B, C and E, while control rabbits without (poly)peptide priming induced antibodies that did not compete with mature b12 and neutralized fewer isolates. The degree of competition with mature b12 for binding to gp140SF162 correlated with the neutralizing activity of the rabbit IgG. Reversing the order of the two boosting immunogens significantly affected the binding profile and neutralization potency of the rabbit IgG. Our study is the first to provide evidence that appears to support the concept that non-HIV immunogens may initiate immune responses leading to elicitation of cross-clade neutralizing antibodies. 3) Long-acting nanoformulated antiretroviral therapy (nanoART) that targets monocyte-macrophages could improve the drug's half-life and protein-binding capacities while facilitating cell and tissue depots. To this end, ART nanoparticles that target the folic acid (FA) receptor and permit cell-based drug depots were examined using pharmacokinetic and pharmacodynamic (PD) tests. FA receptor-targeted poloxamer 407 nanocrystals, containing ritonavir-boosted atazanavir (ATV/r), significantly increased drug bioavailability and PD by five and 100 times, respectively. Drug particles administered to human peripheral blood lymphocyte reconstituted NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice and infected with HIV-1ADA led to ATV/r drug concentrations that paralleled FA receptor beta staining in both the macrophage-rich parafollicular areas of spleen and lymph nodes. Drug levels were higher in these tissues than what could be achieved by either native drug or untargeted nanoART particles. The data also mirrored potent reductions in viral loads, tissue viral RNA and numbers of HIV-1p24+ cells in infected and treated animals. We conclude that FA-P407 coating of ART nanoparticles readily facilitates drug carriage and antiretroviral responses.